Abstract
Cell polarization occurs along a single axis that is generally determined by a spatial cue, yet the underlying mechanism is poorly understood. Using biochemical assays and live-cell imaging, we show that cell polarization to a proper growth site requires activation of Cdc42 by Bud3 in haploid budding yeast. Bud3 catalyzes the release of guanosine diphosphate (GDP) from Cdc42 and elevates intracellular Cdc42-guanosine triphosphate (GTP) levels in cells with inactive Cdc24, which has as of yet been the sole GDP-GTP exchange factor for Cdc42. Cdc42 is activated in two temporal steps in the G1 phase: the first depends on Bud3, whereas subsequent activation depends on Cdc24. Mutational analyses suggest that biphasic activation of Cdc42 in G1 is necessary for assembly of a proper bud site. Biphasic activation of Cdc42 or Rac GTPases may be a general mechanism for spatial cue-directed cell polarization in eukaryotes.